Archive for January, 2011

Making Recombinant Glycosylated Proteins (I)

Many mammalian cell membrane-bound and secreted proteins are glycosylated. The degree of their modifications may be dependent upon tissue specificity and cellular states such as normal vases cancerous. The existence of these proteins in bodily fluids, in combination to their relevance to diseases, makes glycosylated proteins good candidates for clinical diagnostics. Understanding the biogenesis, structure, and functions of these proteins will also aid research and prevention of cancers.

Cancer formation is a heterogeneous and complex process, involving many factors and cellular signaling pathways in each type of cancer. There are more than 1,200 potential cancer biomarkers identified in the literature by a 2006 review. We found that ~ 70% of the 1,261 proteins listed in are naturally secreted proteins, and some 40-50% glycosylated. The addition of carbohydrate groups during protein glycosylation to asparagines (N-linked), or threonines or serines (O-linked) residues may result in mono-, disaccharide- or branched oligosaccharide composed of as many as 20 monosaccharide residues. Glycosylation, together with other modifications, often change the apparent molecular mass of a secreted protein to many folds to that predicted by amino acid sequence. Such heavy modifications on the surface of proteins can influence their functions as well as characteristics as antigens or analytes. Studies of glycosylated proteins offer great opportunities for improving cancer diagnostics.

There are increasing demands for these glycosylated human proteins in good quantity, purity and affordability by the scientific community to perform fundamental and clinical studies in relation to cancer. Such proteins cannot be expressed in bacteria or yeast because those cells do not carry out equivalent post-translation modifications (PTM) as in mammalian cells. Although there have been successful attempts to modify yeast cells to produce proteins with certain types of glycans attached, they were designed for expressing a few pharmaceutical proteins and not suitable for expressing a wide variety of cancer markers. Aside from PTM, expressing human proteins in microorganisms may be hindered by their different codon usage preferences and protein folding tendencies.

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    Wednesday, January 26th, 2011 Uncategorized No Comments

    New SurfaceBind gDNA Isolation and Purification

    Allele Biotech’s SurfaceBind Genomic DNA Pu¬rification Kit is designed for fast, easy, and high-throughput gDNA isolation and purification for lysate obtained through the use of Allele-in-One Mouse Tail Direct Lysis Buffer. Based on our Solid Surface Revers¬ible Binding (SSRB) technology the SurfaceBind system utilizes a plastic tube with its surface coated with proprietary turbo-binders acting to selectively capture and efficiently bind DNA mol¬ecules from reaction mixtures. After lysis of cells, gDNA molecules will specifically interact with the turbo binders and bind to the surface of the tube in the presence of the binding buffer, while pro¬teins and other contaminants will remain in solu¬tion. The DNA can be eluted with as little as 10 microliters of water or buffer for the next application, allowing for a highly concentrated solution.

    The entire process of recovery takes less than 10 minutes with only 1 centrifugation step, making it fast and easy. SSRB technology also provides for maxi¬mum DNA capture and release with limited sam¬ple input, without the DNA loss associated with membrane and bead-based technologies.

    This is a newly developed product particularly for the Allele Biotech’s customers who use the All-in-One mouse tail genotyping kits: get purified genomic DNA using the same lysate you generated for a quick PCR. The yield and purity will enable direct applications to chip assays, sequencing, Southern blotting, etc.Next time you use Allele-in-One Mouse Tail Direct Lysis Buffer be sure to try our SurfaceBind gDNA Purification kit.

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    Wednesday, January 19th, 2011 Customer Feedback, Open Forum No Comments

    Finding the Best Capture Reagents

    As capture reagents, monoclonal antibodies are the most widely used reagents for specifically detecting and quantifying proteins due to their very high specificity. However, development of monoclonal antibodies is time-consuming and expensive. In addition, many antigens prove to be non-immunogenic or extremely toxic, and therefore cannot be used to generate antibodies in animals. Furthermore, the large size of monoclonal antibodies (150 kDa) may limit their use in cases where more than one binding reagent competes for space to recognize closely juxtaposed epitopes. These limitations could arguably be the biggest hurdles to using monoclonal antibodies as capture reagents for a systematic study of the complete human proteome or for clinical applications of advanced proteomics.

    Therefore, alternative capture reagents with high specificity, high affinity, and flexible size and structure that can be easily and cost-effectively produced are urgently needed in order to accelerate proteomic research. Single-chain variable-fragment (scFv) antibodies have been commonly used as alternatives in this regard. scFv is comprised of only the light chain and heavy chain variable regions connected by a peptide linker and with a molecular weight of 27 kDa. Since scFv retains the antigen-binding site of the variable regions, it inherits the specificity of an intact antibody and affinity. In addition, scFv can be easily expressed in yeast or in E. coli with yields in milligrams per liter. scFv can be linked to Fc of desired species specificity and maintain binding properties. If necessary, there is also the option of converting scFv into other antibody formats such as Fab or full IgG by simple cloning steps. The converted antibodies can also be efficiently expressed and purified in yeast or E. coli.

    More recently, single domain antibodies that exist in nature were discovered that can be as small as half the size of scFv, and judging from the available data, superior in binding capabilities to scFv or even traditional IgG antibodies. This type of affinity molecules, termed VHH isolated from camelid animals or nurse shark, can be highly expressed in E. coli, linked to a fluorescent protein marker, or chemically conjugated to HRP or other signal generating moieties through a one step reaction.

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    New Frontiers for Research Tool Development in the New Year

    Looking into the future of technologies in biology research

    Allele Biotech's Green Crystal Ball

    Chosen as the Method of the Year 2010 by Nature Method and mentioned in a number of year-end recaps, this is a technology that allows the use of light to precisely (at least in a temporal sense) control engineered proteins within a targeted cell population. For example, by introducing light-activated channelrhodopsins into neurons, one can use a pulse of light to initiate a movement of ion across the cell membrane. The technology, first reported in 2005 then made headlines as a major impact on neurosciences since 2007, is now being combined with other components in controlling a broader array of biological events, such as DNA binding, enzyme activities, etc. Looking forward, a few areas will be more than likely the frontlines of moving optogenetics into more labs:

    Additional combinations: The few known channelrhodopsins and their fast growing variations will be combined with more “effecter” domains to control different events. The challenge will be to find ways to use the structural changes or any responses channelrhodopsins have to stimulating lights in order to trigger a reaction in the associated effecter domain.

    Tracking mechanisms: A platter of fluorescent proteins (FPs) will be used as an independent tracking method to follow cells being targeted. FPs that have optical spectra that do not interfere with the optogenetic molecules will be tested and established. In addition, FPs with less toxicity, narrower excitation and emission peaks, and more tolerance to different cellular environment will be preferred and eventually set up as standards.

    Delivery tools: To bring the optogenetic reagents into cells like neurons researchers will most likely rely on lentiviral vectors in most cases. Other vehicles such as baculovirus, MMLV-based retrovirus, even herpes virus may find broader applications in this field. Pre-packaged lentiviruses and MMLV-retroviruses already contain optogenetic constructs will become popular products.

    VHH Antibodies
    The small capture polypeptides based on single-domain Camelid antibodies (nanobodies, nano antbodies or nAbs) and similar VHH domains will become much dramatically more popular this year, judging from the significant increase in demands of the only camelid reagent products, GFP-Trap and RFP-Trap, in 2010. There are a number of NIH initiated programs that aim to find capture reagents that eventually target the complete human proteome. One of the key criteria for the current phase of the relevant NIH Director’s Initiative is ability to co-immunoprecipitate. The Human Proteome Organization (HUPO) recently expressed frustration due to the lack of high quality capture reagents necessary to isolate and identify most proteins. HUPO promotes global research on proteins in order to decode the human proteome. From what we have learned from dozens of publications showing the use of GFP-Trap, VHH molecules pulls down GFP-tagged proteins with unprecedented efficiency and purity. VHH antibodies show strong affinity and specificity, at a level superior or comparable to monoclonal antibodies. In addition, VHH antibodies are increasingly appreciated for their capabilities to recognize concave epitopes by their relatively convex-shaped paratopes. VHH nanobodies are small (~12-15 kD), with a limited number of functionally important disulfide bonds, can be expressed very well in E. coli, and are amazingly stable in extreme denaturing conditions such as heat and acid. They have been shown to be better suited for in vivo and trans-cellular membrane delivery than other antibodies. It should not be surprising that one day in the coming years VHH antibodies will be more dominant than monoclonal antibodies.

    Super-Resolution Imaging
    One of the goals of developing technologies such as photoactivated localization microscopy (PALM) and related super-resolution imaging (SRI) techniques was to achieve electron microscopy (EM) level resolution without using EM. Now new developments show that maybe combining EM and photoactivable FPs would provide more specific and more detailed morphology. It would be anticipated that more photoconvertible FPs will prove to work well for one type of SRI or another. The event that will bring this technology to nearly every cell biology lab is the improvement and availability of necessary instruments that some companies have already begun to commercialize.

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